This project is concerned with the role of the enzymes involved in pyruvate metabolism in genetic diseases. The specific enzymes involved are: pyruvate dehydrogenase complex, pyruvate carboxylase and P-enolpyruvate carboxykinase. Part of the project deals with the elucidation of structural, kinetic, regulatory and mechanistic properties of these enzymes. A second protion of the project attempts to use this knowledge to develop appropriate assays for the detection of genetic diseases related to these enzymes and to develop methods for evaluating the nature and extent of the physiological effects of the disease in vivo. Present plans call for the continued application of methods already developed for these three enzymes to cultured skin fibroblasts to patients with lactic acidoses and neurological and developmental deficits of unknown etiology. Other studies will consider the significance of multi-site phosphorylation in the regulation of pyruvate dehydrogenase complex in animal mitochondria and the possible relationship of this process to diseases involving pyruvate dehydrogenase complex. Finally, deuterated glucose and (13C)-aminobutyrate will be used to measure glucose turnover and pyruvate oxidation in patients with suspected abnormalities in these metabolic areas.